This research project deals with searches for unknown intracellular molecules involved in the regulation of autophagy, which contributes to bulk protein degradation in the cell. Results obtained through the project for these 2 years are as follows :1. Search for possible messenger molecules involved in amino acid signaling : Because of the importance of the quality of S.aureus alpha-toxin as a permeabilizing agent for the bioassay system, the method for culturing the bacteria and purifying the toxin was improved. The low MW extract of the liver stimulated by regulatory amino acids exhibited an inhibitory effect on autophagic proteolysis in alpha-toxin permeabilized hepatocytes. The active fraction was stable with heat, and stable at acidic and neutral pH, but unstable at alkaline pH, showing a positive charge. The possiblity that it may act on the autophagosome formation step which is regarded as a target site of amino acid action was indicated.2. Identification of the protein phosphor
… Moreylated by glucagon : In order to know the regulatory mechanism by glucagon, isolated rat hepatocytes was stimulated by glucagon with ^<32>Pi. A 49 kDa protein phosphorylated by glucagon in vivo was discovered in the crude lysosomal fraction, It was confirmed that it was phosphorylated by cAMP through protein kinase A in vitro.3. Maturation step of autophagy and GTP binding protein(s) : The permeabilized hepatocytes by streptolysin 0 (SLO), an another pore-forming toxin, was employed to clarify the maturation step of autophagy. From inhibitor studies, the involvement of some GTP binding proteins and an NEM sensitive protein was indicated at the maturation step. During the project, troubles with the potency of SLO took place, making it difficult to continue the assay. Thus, as an alternative method, an membranefusion assay between autophagosome and lysosome, an in vitro system, is now tried to be established. A cytosolic enzyme, betaine-homocystein methyltransferase, of which limited proteolytic intermediates after autophagic fusion can be identified immunochemically, is expected to make use as a probe for the fusion step. Less